The present invention relates to optical transmission measuring cells and reactors with an integrated optical detection mechanism and, especially, to a micromechanical transmission measuring cell for determining an optical absorption of a sample fluid.
Reactors with an without integrated evaluation components are presently used in various fields of analytical and synthetic chemistry. One embodiment which is frequently used in the field of analytical chemistry is the microtitration plate, which is used for immunological testing methods, e.g. the enzyme-linked immunosorbent assay (ELISA). Microtitration plates normally consist of an optically transparent plastic body provided with a number of depressions used as reaction vessels. The inner wall of said reaction vessels is coated with a suitable biochemical receptor layer which, when the sample fluid has been filled in, permits selective bonding of the analyte molecule to be determined to at least one reactor wall. In further reaction steps, a change in colour is produced in the reaction vessel as an indicator reaction, said change in colour representing a measurable variable for the amount of bonded analyte molecules. The quantitative determination of the alteration of colour is normally carried out by an optical transmission measurement through the interior volume of the reactor and through the plastic body.
Further embodiments of microreactors consist of a volume through which a flow passes, e.g. a capillary or a flow passage filled with a carrier material, on the inner surface of which (the inner wall or surface of the carrier material) a receptor layer is immobilized. Such a system is described in E. Yacoub-George, H. Wolf, S. Koch, P. Woias, A Miniaturized ISFET-ELISA System with a Pretreated Fused Silica Capillary as Reaction Cartridge, Proc. of the Transducers ""95xe2x80x94Eurosensors IX, Stockholm, Sweden, 1995, pp. 898-901. The chemical reaction mechanism used in this case is similar to the above-described course of action and produces as the last step again an indicator reaction in the volume of fluid contained in the reactor. The inner volume of the reactor is then supplied to a subsequent evaluation component, e.g. a photometer or an electrochemical sensor, for carrying out the quantitative determination of the indicator reaction.
Like reactors with an without integrated evaluation components, also a great variety of optical transmission cells is presently used in the field of analytical and synthetic chemistry. Simple embodiments consist of measuring cuvettes which are filled with the liquid to be analyzed and introduced in the ray path of an array comprising a light source and an optical detector. Flow-through cuvettes, however, comprise a flow passage which is introduced in the ray path of the optical array in the direction of flow or transversely to the direction of flow, said optical array consisting of a light source and of an optical detector.
E. Verpoorte, A. Manz, H. Lxc3xcdli, H. M. Widmer, B. H. van der Schoot, N. F. de Rooij, A Novel Optical Detector for Use in Miniaturized Total Chemical Analysis Systems, Transducers ""91, Book of Abstracts, pp. 796-799, describes a micromechanical flow-through cuvette, which consists of a channel realized by means of anisotropic etching processes and covered on the upper surface thereof by a silicon chip provided with windows. Due to the use of silicon wafers with an  less than 100 greater than  crystal orientation, the lateral walls of the channel, which have been produced by anisotropic etching, have the orientation of the etch-resistant  less than 111 greater than  crystal plane. As is known to those skilled in the art, this plane extends at an angle of approx. 54xc2x0 to a horizontal reference plane. In the known micromechanical flow-through cuvette, light is coupled in by being radiated in approximately perpendicularly through an optical entrance window by means of a light waveguide, which is directed onto an inclined end face of the etched channel. Perpendicularly means in this context a direction perpendicular to the direction of flow of the sample fluid. Due to the reflection at one end face of the channel, light is guided into the cell interior and, due to multiple reflections on the lateral walls, it is guided to the second end face, where it is coupled out of the channel through an optical window, i.e. through the cover chip, and fed into a glass fibre arranged at right angles to the direction of flow of the sample fluid. It follows that the light is coupled out at the second end face, the outcoupling glass fibre leading to a detector which may have a conventional structural design.
One disadvantage of commercially available microtitration plates is that they have typical inner volumes of the reactor in the range of some ml and diffusion path lengths in the range of some mm. This has the effect that the development of the chemical processes in the interior of the reactor, i.e. the bonding of the analyte molecules to the receptor layer, the generation of the indicator value, etc., is mainly determined by the comparatively long diffusion paths and the resultant long diffusion times. The time required for an analysis can therefore be in the range of some hours.
Furthermore, microtitration-plate tests are processed by automated anlysis apparatus which must have a comparatively high mechanization degree (e.g. pipetting robots, plate transport mechanisms), whereby the costs and the error rate are increased.
The use of reactors without integrated evaluation components normally requires additional transport steps at the end of the indicator reaction, and these additional transport steps may result in a higher expenditure and, depending on the respective structural design, in signal losses, e.g. due to mixing processes during transport in a flow-through system.
Optical transmission cells operating according to the cuvette principle have a comparatively large fluid volume in the range of some ml and are not suitable for flow-through operation. It follows that automatic processing of sample series can only be carried out with a high mechanical outlay making use of a robot system or of automatic handling machines.
Optical flow-through cuvettes are often produced by conventional techniques, e.g. injection moulding of plastic material, whereby miniaturization is only possible to a certain degree.
The micromechanical flow-through cuvette in silicon technology, which has been mentioned hereinbefore, additionally has, as has been described hereinbefore, an intentionally chosen perpendicular coupling-in direction of the light so that a ray path with multiple reflections on the channel walls is obtained. The large number of multiple reflections is intended to substantially increase the effective optical path length of the cell in comparison with the channel diameter by factors of 10 to 50, whereby an improved detection sensitivity is to be achieved. Since the etched silicon channel walls permit, however, only lossy reflections, a high light power must be coupled into this known transmission measuring cell so that a measurable light power can be coupled out at the outlet.
U.S Pat. No. 4,908,112 discloses a silicon semiconductor wafer for analyzing micronic biological samples. The analytical device comprises a separation channel having an elongate shape, electrodes formed in said channel, and a storage reservoir as well as a reception reservoir. The single separation channel or a plurality of separation channels are formed in a silicon wafer and have bevelled walls which are typically produced when etching by means of a potassium hydroxide solution is carried out. A silicon dioxide layer is formed on the channel. The electrodes are used for activating a movement of the sample fluids through said channel by means of electroosmosis. For analyzing the sample, a laser beam is directed onto a bevelled side wall of the channel; from said bevelled side wall, the laser beam is reflected transversely across the channel transversely to the opposite wall, and from said opposite wall it is reflected out of the channel. By means of the incident laser light, fluorescence is produced in a suitable analytical sample, the fluorescent light emerging from the channel being detected by means of a photodetector. The fluorescent light is used for determining the components of the sample which have been separated by electromigration.
EP 0 488 947 Al discloses a miniaturized detector cell produced from silicon or quartz. Said detector cell comprises a channel in which a sample fluid is contained as well as an inlet and an outlet window for light for carrying out a transmission measurement. The light is radiated perpendicularly onto an inclined wall of the channel so as to carry out multiple reflections in the channel and in the elongate receptacle, respectively; subsequently, it is directed from the outlet opening to a suitable detector. These multiple reflections increase the length of the interaction path substantially.
DE 41 37 060 A1 discloses a microcuvette for infrared spectroscopy. Said microcuvette comprises a channel and inlet and outlet openings for a sample fluid, which are arranged at right angles to said channel. Light is radiated in directly through silicon discs defining the cuvette.
GB 2 071 355 A discloses a fluid cell which is used for a spectroscopic analysis and which comprises a front plate as an optical window, a rear plate having a reflecting surface, and a sealing means between these two plates. The rear plate is provided with an inlet and an outlet opening for fluids which are to be analyzed in said cell.
Reference 6 (CH 674 082 A5) discloses a cuvette comprising as a main element a prism with a chosen form which consists of a transparent material and the refractive index of which is higher than the refractive index of the solution to be examined. A cavity filled with a fluid to be analyzed is arranged above said prism, where as an empty cavity is located below said prism. In this cuvette, the light propagates substantially in said prism and is influenced by a fluid arranged in side-by-side relation with said prism.
It is the object of the present invention to provide a micromechanical transmission measuring cell which permits a low consumption of sample fluid and reagents and which achieve s a high detection accuracy.
This object is achieved by a micromechanical transmission measuring cell according to claim 1.
The present invention is based on the finding that, for a high detection accuracy, light, which has been coupled into a sample fluid receptacle, must be prevented from being subjected to multiple reflections on the inner wall of the receptacle so as to limit the reflection losses in the sample fluid receptacle to a minimum. Hence, a bundle of quasi-parallel rays, which will be referred to as xe2x80x9clight beamxe2x80x9d in the following, is used for transmission measurement in the micromechanical transmission cell according to the present invention, the diameter of said xe2x80x9clight beamxe2x80x9d being smaller than the internal cross-section of the fluid receptacle. This xe2x80x9clight beamxe2x80x9d is coupled in onto a first reflector in a defined manner in such a way that it passes through the sample fluid receptacle, which can be produced micromechanically in a silicon substrate, largely without any multiple reflections on the receptacle wall. This reflector can be coated e.g. with gold, whereby its reflective properties are optimized. It is not necessary to coat the rest of the inner wall with gold, since only a small amount of the light fed into the receptacle is subjected to multiple reflections on the receptacle wall.
The light used for the measurement may not normally be present in the form of a xe2x80x9clight beamxe2x80x9d, but it may be divergent. In such cases, a collimating lens system can optionally be inserted in the light ray path at a suitable location. This method is generally known from the field of optics. When the divergent light emerging from a light waveguide is used, it will be expedient to apply a gradient lens of suitable length directly onto the end face of the light waveguide.
The micromechanical transmission cell for determining the optical absorption of a sample fluid comprises a receptacle for holding the sample fluid, a light passage opening for introducing the light into the receptacle, and the reflector directing the light relative to the receptacle in such a way that most of said light passes through said receptacle without multiple reflections on a wall of said receptacle.
In comparison with macroscopic reactors, such as the microtitration plate described at the beginning, the micromechanical transmission cell according to the present invention offers the advantage of a small internal volume of the reactor, said advantage being possible due to the micromechanical silicon processing technique. This results in short diffusion paths and diffusion times and in a low consumption of reagents, analyte and sample fluid. The micromechanical transmission cell can be provided with a sample-fluid inlet opening through which a sample fluid is introduced in the receptacle, whereupon said micromechanical transmission cell is operated in the so-called xe2x80x9cstopped-flowxe2x80x9d mode, i.e. the sample fluid does not flow through said micromechanical transmission cell but it stands still so to speak in said cell. By providing a sample-fluid outlet opening, the micromechanical transmission cell according to the present invention can, however, also be used in a flow-through mode. Hence, it is adapted to be used in a very flexible manner. When an inlet and an outlet opening are provided, xe2x80x9cstopped-flowxe2x80x9d operation is possible as well. In this case, the reagent is pumped in, whereupon the pump is stopped and the reaction is allowed to take place.
In comparison with reactors without an integrated evaluation component (such as a fused silica capillary), the micromechanical transmission cell permits an in-situ determination of reaction results without additional transports being necessary. Furthermore, reaction developments in the interior volume of the reactor can be determined in situ e.g. by measurements of the reaction kinetics.
As has already been mentioned, the main advantages which the micromechanical transmission cell shows in comparison with the known micromechanical flow-through cuvette including an integrated evaluation component are the minimum amount of reflections in the sample-fluid receptacle resulting from the fact that the ray path is chosen such that it extends parallel or perpendicular to the direction of flow as well as parallel to the receptacle wall, whereby reflection losses of the light transmitted through the receptacle are minimized. Furthermore, the micromechanical transmission cell according to the present invention permits arbitrary combination possibilities of coupling the light in and out and of supplying and discharging the fluid on the upper and on the lower side of the receptacle.
By immobilizing on the inner wall of the receptacle a biochemical component which initiates or influences a chemical reaction in the reactor interior by interaction with an associated reaction partner to be detected, e.g. an enzyme substrate or an antigen, and which produces in this way an optically detectable reaction result that can be correlated with the analyte concentration, the micromechanical transmission cell according to the present invention can also be used as a biochemical reactor, without the transmission being influenced by the reflective properties of the biochemical component on the inner wall of the receptacle. Without immobilizing a biochemical component on the receptacle, the micromechanical transmission cell can be used as a universally usable transmission cell.